Unwanted T cell activation is known to be associated with a number of pathological, immunological disorders such as, for example, autoimmune diseases, allergic responses and transplant rejections. Autoimmune diseases are a particularly important class of the diseases involving deleterious or unwanted immune responses. In autoimmune diseases, self-tolerance is lost and thus, the immune system attacks "self" tissue as if it were a foreign target. More than 30 autoimmune diseases are presently known to exist; myasthenia gravis (MG) and multiple sclerosis (MS), for example, are two autoimmune diseases which have received wide-spread public attention.
Moreover, a number of allergic diseases have been found to be associated with particular MHC alleles or have been suspected of having an autoimmune component. Additionally, other deleterious T cell-mediated responses include the destruction of foreign cells that are purposely introduced into the body as grafts or transplants from allogeneic hosts. This process, known as "allograft rejection," involves the interaction of host T cells with foreign MHC molecules. Quite frequently, a broad range of MHC alleles are involved in the response of the host T cell to an allograft.
The current model of immunity postulates that antigens mobilize an immune response, at least in part, by being ingested by an antigen-presenting cell (APC) which contains on its surface a MHC Class II glycoprotein encoded by a gene in the major histocompatibility complex (MHC). The antigen is then presented to a specific T helper cell in the context of the surface bound MHC glycoprotein. By interaction of the antigen specific T cell receptor with the antigen-MHC complex, the T helper cell is stimulated to mediate the antigen-specific immune response, including the induction of cytotoxic T cell function, the induction of B cell function, and the secretion of a number of factors aiding and abetting this response.
The involvement of the MHC Class II proteins in autoimmune diseases has been shown in animal models. Administration of antibodies either to the MHC Class II proteins themselves or to the agents that induce expression of the MHC Class II genes interferes with the development of the autoimmune condition in these model systems. The role of T helper cells has also been demonstrated in these models by counteracting the autoimmune system using anti-CD4 monoclonal antibodies; CD4 is the characteristic T helper cell receptor (Shizuru, et al., Science 240:659-662 (1988)).
Recent experiments have shown that, under certain circumstances, anergy or nonresponsiveness can be induced in autoreactive lymphocytes (See, e.g., Schwartz, Cell 1073-1081 (1989), which is incorporated herein by reference). In vitro experiments suggest that antigen presentation by MHC Class II molecules in the absence of a co-stimulatory signal induces a state of proliferative non-responsiveness in syngeneic T cells (Quill, et al., J. Immunol. 138:3704-3712 (1987), which is incorporated herein by reference). These reports, however, provide no clear evidence that induction of anergy in vivo is possible or that autoimmune disease can be effectively treated in this manner.
More recently, complexes and methods have been described that are useful for identifying and inhibiting those aspects of the immune system that are responsible for undesirable immune responses, such as, for example, autoimmunity. See, U.S. Pat. Nos. 5,130,297 and 5,194,425, both of which are incorporated herein by reference. These complexes and methods are designed to target T helper cells which recognize a particular antigen in association with a glycoprotein encoded by the MHC. The complexes effectively bind T cell receptors and cause non-responsiveness in target T-lymphocytes and other cells of the immune system.
These complexes, which are useful for modulating T cell function, consist of (1) an effective portion of the MHC-encoded antigen-presenting glycoprotein; and (2) a peptide representing a fragment of an autoantigen or other antigenic sequence associated with the disease state to be treated (i.e., an antigenic peptide). The association between the antigenic peptide and the MHC glycoprotein can be by covalent or noncovalent bonding. Evidence from both in vitro and in vivo experiments unequivocally establishes that such complexes induce clonal anergy in syngeneic T cells.
The MHC component of these complexes is typically prepared by purification from cell surfaces. However, purified MHC molecules isolated from cell surfaces usually contain prebound endogenous peptides. (See, Chicz, et al., Nature 358:764-768 (1992) which is incorporated herein by reference.) When using MHC-peptide complexes to modulate T-cell function, it is desirable to use a composition of homogeneous complexes of an MHC molecule and a defined antigenic peptide entity. A major limitation in obtaining such a composition lies in the inability of the previously used purification method to successfully separate the MHC-peptide complexes of interest from both uncomplexed MHC molecules and other endogenous MHC-peptide complexes.
To date, the only method used to separate the MHC class II-peptide complexes of interest from free MHC molecules or endogenous peptide bound-MHC complexes involves the use of a biotin-avidin system. (See, Demotz, et al., Proc. Nat'l Acad. Sci. U.S.A. 88:8730-8734, which is incorporated herein by reference.) This method has limitations in the sense that it involves modification of the peptide component in order to prevent side reactions during biotinylation. Moreover, using this method, recovery of the purified product can vary significantly, and the purified complexes have been found to have some residual contaminations. To overcome this latter problem, another anti-hapten antibody affinity chromatography was added prior to the avidin column chromatography. This additional step, involving the addition of a DNP ligand to the peptide, reduces the recovery of the purified complexes even further to only about 0.4 to 4 percent of the starting complexes.
Thus, there still remains a need for purification methods that are easy to carry out, readily scalable and wherein the recovery of the purified MHC-peptide complexes is relatively high.